The aim of this study was to determine whether maternal serum matrix metalloproteinases 2, 3, 9, and 13 levels differ in early- and late-onset preeclampsia and uncomplicated pregnancies. The study was carried out in 125 pregnant women . Levels of MMP-2, MMP-3, MMP-9, and MMP-13 were measured in the maternal serum using an enzyme-linked immunosorbent assay. Maternal serum MMP-2 levels in both the groups of preeclamptic women were significantly higher than those in the controls. Levels of MMP-3 were significantly higher in preeclamptic patients with early-onset disease; however, the MMP-3 levels in patients with late-onset preeclampsia were similar to those observed in the control subjects. MMP-9 levels were lower whereas the levels of MMP-13 were higher in both preeclamptic groups of pregnant women than in the healthy controls, but these differences were statistically insignificant. One important finding of the present study was that MMP-3 appears to be involved solely in early-onset preeclampsia, but not in late-onset preeclampsia. Higher levels of MMP-2 and MMP-13 and lower levels of MMP-9 seem to be related to both early- and late-onset severe preeclampsia.  Although there have been extensive studies of preeclampsia, its aetiology remains unclear –6. HowevReister et al.  presenteMetalloproteinases zinc-dependent enzymes have been reported to be involved in extracellular matrix remodelling and processes of placental angiogenesis with structural spiral arteries transformation, which precedes proper trophoblast invasion , 6, 10. The increase of both the local expression and activity of some metalloproteinases was observed in pathologically changed human arteries and atherosclerotic plaques. Two gelatinases: MMP-2 and MMP-9, stromelisyn-1 (MMP-3), and collagenase MMP-13 belong to a group of metalloproteinases directly linked to the process of blood vessels remodelling . The keyThe results of previous research into the role and expression pattern of metalloproteinases in pregnancies complicated by preeclampsia are not unambiguous.Therefore, the aim of this study was to explore whether severe preeclampsia is associated with changed maternal serum matrix metalloproteinases MMP-2, MMP-3, MMP-9, and MMP-13 levels .The patients who had been referred to the tertiary-level academic unit for further treatment and perinatal surveillance because of preeclampsia without any signs of labour were offered participation in this study. Informed consent was obtained from each woman for the participation in the study and for peripheral blood sampling and the protocol for the study was approved by the Bioethical Board of the Medical University of LublinA total of 60 pregnant women at 26–38 weeks of gestation, with pregnancies complicated by severe preeclampsia (29 women with early-onset preeclampsia and 31 women with late-onset preeclampsia) and 65 pregnant women with normotensive, uncomplicated third-trimester pregnancies were included in the study. Blood pressure had been measured on each pregnant patient's arm supported and positioned at the level of her heart on two occasions at least 4 hours apart, with using the first Korotkoff sound for evaluation of the systolic blood pressure and the fifth for assessment of the diastolic blood pressure [Severe hypertension was defined as systolic blood pressure ≥ 160 mmHg and diastolic blood pressure ≥ 110 mmHg.Severe preeclampsia was defined according to new American College of Obstetricians and Gynecologists' guidelines as new-onset hypertension in the second half of pregnancy accompanied by new-onset proteinuria and/or haematologic abnormalities , cerebral or visual disturbances, renal or liver impaired function, HELLP syndrome , or pulmonary oedema . Early-oNone of the preeclamptic pregnant patients had been affected by chronic hypertension, renal disorders, or proteinuria before pregnancy and all of them were normotensive before the 20th week of gestation. All preeclamptic women were normotensive 3 months after labour .None of the 65 healthy controls had signs of elevated blood pressure or other pregnancy disorders or complications and all of them gave birth to healthy infants. None of the patients from this group suffered from proteinuria. All patients in the study were nonsmokers.All women were with singleton pregnancies. All cases of premature rupture of membranes, chorioamnionitis, inflammatory conditions, and other medical diseases such as chronic hypertension, diabetes mellitus, autoimmune disorders, and heart, renal, or liver diseases prior to the pregnancy were excluded from the study.Upon admission data were collected concerning maternal anthropometric parameters and medical history was taken. In all patients, blood was drawn for assessment of serum levels of matrix metalloproteinases MMP-2, MMP-3, MMP-9, and MMP-13. Ten millilitres of blood was collected by venipuncture from each preeclamptic woman and from each woman in the control group before any drug administration. The blood samples were placed in sterile tubes at 4°C and centrifuged for 15 min at 1500 ×g. Afterwards, the serum samples were stored at –70°C until assayed.The levels of maternal serum MMP-2, MMP-3, MMP-9, and MMP-13 were determined using a specific and sensitive commercially available enzyme-linked immunosorbent assays (ELISA assays) according to the manufacturer's instructions .For MMP-2 a minimum detectable level was 0.047 ng/mL with intra-assay 5.5% and interassay 6.9% precision rate. For MMP-3 a minimum detectable level was 0.3 ng/mL with intra-assay 7.3% and interassay 8.8% precision rate. For MMP-9, the lowest detectable concentration was 0.5 ng/mL and for MMP-2 the minimum detectable level was 0.047 ng/mL with intra-assay 7.3% and interassay 10.2% precision rate. For MMP-13 the minimum detectable level was 0.18 ng/mL with intra-assay 7.4% and interassay 8.2% precision. t-tests, or  the Cochran-Cox test.In the statistical analysis, results were expressed as mean ± SD or SEM or as median values and were statistically analysed with the computer program “Statistica 8” using the Shapiro-Wilk test for the normally distributed data and equality of variance by Levene test and, subsequently, two-tailedp value of less than 0.05 was considered to be significant.The ANOVA and Kruskal-Wallis tests were used to test for differences among 3 independent groups. A statistically significant effect in ANOVA was followed by follow-up post hoc Tukey's test in order to assess which group is different. A In the present study 125 pregnant women at 26–38 weeks of gestation had been investigated. Sixty patients had a diagnosis of severe preeclampsia and were divided into two study groups with early- and late-onset disease (the earlyPre and the latePre groups), and sixty-five of them had healthy normotensive pregnancies without any complications (the control group).The general demographic characteristics and obstetric history of the study population are shown in In the present study it was found that both groups of preeclamptic patients with early- and late-onset preeclampsia had significantly higher MMP-2 levels than the healthy controls and were distributed as follows: 242.93  ±  68.64 ng/mL (136.88–466.90 ng/mL) in earlyPre, 234.39.7 ± 79.18 ng/mL (124.19–514.79 ng/mL) in late-onset preeclampsia, and 195.30 ± 43.53 ng/mL (103.88–347.83 ng/mL) in the control group.One important finding of the present study was that MMP-3 levels were significantly higher in the earlyPre group than in the preeclamptic patients with late-onset preeclampsia (latePre group) and the controls. There were no differences in MMP-3 levels between the group of patients with pregnancy complicated by late-onset preeclampsia and the control subjects.To the best of my knowledge, this is the first study that has detected high levels of MMP-3 in patients with early-onset preeclampsia and unaltered levels of MMP-3 in patients with late-onset preeclampsia.The mean values were 63.54 ± 71.58 ng/mL (13.65–314.14 ng/mL) in the earlyPre group and 27.91 ± 24.99 ng/mL (4.16–124.17 ng/mL) in the latePre group compared with 29.31 ± 58.79 ng/mL (2.046–454.53 ng/mL) in the control group.Maternal serum levels of MMP-9 were lower in patients with pregnancies complicated by preeclampsia. The levels of MMP-13 were higher in preeclamptic women, especially in patients with early-onset preeclampsia. But these differences were statistically insignificant. The mean values of MMP-9 and MMP-13 were 1450.12 ± 838.01 ng/mL (243.68–3383.13 ng/mL) and 0.590 ± 0.681 ng/mL (0.054–4.351 ng/mL) in the earlyPre group, 1450.86 ± 1165.93 ng/mL 325.07–5430.0 ng/mL) and 0.482 ± 0.278 (0.008–1.264 ng/mL) in the latePre group, and 1725.53 ± 901.54 ng/mL (336.83–4569.2 ng/mL) and 0.427 ± 0.228 ng/mL (0.014–1.047 ng/mL) in the healthy controls, respectively.The present study revealed higher levels of MMP-2 and MMP-13 in both groups of the studied patients with early- and late-onset preeclampsia in comparison with healthy normotensive controls. The levels of MMP-9 were lower in the patients with pregnancy complicated by severe preeclampsia. MMP-3 levels were significantly higher in the patients with early-onset preeclampsia than in the late-onset preeclampsia and the controls. There were no differences in MMP-3 levels between the patients with pregnancy complicated by late-onset preeclampsia and the control subjects.Similar results of higher levels of MMP-2 and lower levels of MMP-9 in maternal serum of preeclamptic women were found by Montagnana et al. . These aPalei et al. observed increased activity of MMP-9 and MMP-9/TIMP-1 ratio in pregnancies complicated by gestational hypertension, but not in those with preeclampsia whereas MMP-2 did not show any differences with respect to healthy pregnancies and pregnancies complicated by hypertension or preeclampsia .However, our studies showed different results.Although Gerlach et al.  do not rHowever, taking into account the lower serum MMP-9 levels in preeclamptic pregnancies obtained in our study, it can be supposed that the plasma MMP-9 levels in these women would be even lower, which further confirms our findings. For our defence we can assure that all collected samples of serum were prepared and frozen at a temperature of 70°C to an analysis directly after taking blood, in order to avoid elevating of MMP-9 levels artificially.Reiter indicates that impaired formation of uteroplacental arteries is associated with an increased risk of preeclampsia . It has However, it is difficult to propose possible clinical application of tissue inhibitors of metalloproteinases (TIMPs) in treatment, since some reports reveal that TIMP-2 are capable of blocking the vasodilator effect of MMP-2 in small blood vessels, which additionally would potentiate pathological mechanism observed in preeclampsia –27. MoreAt the same time, results of the present study can suggest the importance of increased MMP-2 levels as a defence mechanism against increased vasoconstriction observed in small prearteriolar vessels in pregnancy complicated by preeclampsia .However, different results were presented by Palei et al., who did not observe changes in MMP-2 activity neither in pregnancies complicated by gestational hypertension nor by preeclampsia with respect to healthy pregnancies .Despite initially promising signals about possible use of MMPs inhibitors, it is hard to clearly determine their role in medical treatment –27. StudMMP-9 is known to participate in trophoblast cells invasion and to be involved in the formation of new blood vessels and thus it is called a trigger of angiogenesis . In addiCoolman et al.  put forwIn view of the literature data, the results of the present study may suggest the importance of lower maternal serum MMP-9 levels in abnormal development of blood vessels at the interface between mother and fetus in pregnancies complicated by severe preeclampsia. Higher levels of MMP-2 seem to be responsible for the endothelial damage and severe vasoconstriction observed in severe preeclampsia. At the same time, taking into account suggestions that MMP-2 is taking part in process of vasodilatation, its elevated concentrations could constitute an attempt to compensate alterations observed in pregnancies complicated by preeclampsia.Different results were also presented by Poon et al. , who obsOne new and important finding of the present study was that MMP-3 levels were significantly higher in the earlyPre group than in the preeclamptic patients with late-onset preeclampsia (latePre group) and the controls.MMP-3 is also referred to as stromelysin-1. It digests extracellular matrix components, can activate the latent forms of matrix metalloproteinases, and may induce apoptosis . MMP-3 pMechanisms responsible for controlling trophoblast cells mobility are considered to stimulate production and secretion of MMP-3. On the other hand, inappropriate activation of MMP-3 in the presence of cytokines Th1 such as Il-1b is likely to influence the development of pregnancy complications.Husslein et al. put forward that active MMP-3 is produced in villous and extravillous trophoblasts of early pregnancy and decreased expression of MMP-3 was observed in later gestation .On the other hand, Reister et al.  found stIt was observed that MMP-3 contributes to trophoblast motility and plays an important role in physiological remodelling processes  and tropThe aforementioned data from literature and the findings of other researchers along with the results of the present study seem to point to the importance of higher levels of MMP-3 in the development of early-onset preeclampsia, but not in late-onset form of preeclampsia.It is also feasible that higher maternal levels of MMP-3 in early-onset preeclampsia may cause the disease or ability of compensation for occurring disturbances.Elevated concentrations of MMP-3 in pregnancies complicated by early-onset preeclampsia and nonaltered ones in pregnancies complicated by late-onset preeclampsia can also indicate the importance of the process of altered trophoblast invasion for the early subtype of the disorder, sometimes called the fetal type, and a less significant role of MMP-3 in late-onset preeclampsia, called well the maternal type, where clinical symptoms appear later. Therefore, it may suggest lower significance of this metalloproteinase in maternal cardiovascular changes and higher significance for placental vessels, in maternal-placental-fetal unit.Higher levels of MMP-2 and MMP-13 and lower levels of MMP-9 seem to be related to both early- and late-onset preeclampsia.It seems that proper regulation of metalloproteinases activity is essential to maintain homeostasis of extracellular matrix, and the imbalance of expression and activity of MMPs leads to pathological changes and disorders.In summary, the findings of the present study suggest the importance of metalloproteinases in aetiopathogenesis of both early- and late-onset severe preeclampsia. Early- and late-onset severe preeclampsia seem to be associated with increased maternal serum levels of MMP-2 and MMP-13 and decreased levels of MMP-9.It seems that lower maternal serum MMP-9 levels may lead to abnormal development of blood vessels at the interface between mother and fetus in preeclamptic pregnancies.Increased MMP-2 levels may reflect ischemic disturbances and vascular endothelial dysfunction in pregnancies complicated by severe preeclampsia or could constitute an attempt to compensate alterations observed in preeclamptic pregnancies.Furthermore, MMP-3 appears to be involved solely in early-onset preeclampsia. To the best of my knowledge, this is the first study to detect higher maternal serum levels of MMP-3 in patients with early-onset preeclampsia and unaltered levels of MMP-3 in patients with late-onset preeclampsia. These results can also indicate the importance of altered trophoblast invasion only for the early subtype of the disorder.The potential limitation of the present study is mainly related to the small sample size and the fact that only levels of MMPs were evaluated. The enzymatic activity of the MMPs would be justified to be assessed in order to confirm that higher levels of metalloproteinases are linked to their higher enzymatic activity. The precise determination of a potential role of MMPs in preeclampsia requires further investigation with the application of a larger group of patients.


Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with  Epidemiological studies have demonstrated an association between the consumption of tomato products and the carotenoid lycopene and a decreased risk of prostate cancer ,2, a rel14C-labeling was used to study the biodistribution of lycopene in rodents [14C is radioactive, the amount of 14C-lycopene that can be safely administered is limited and cannot be used to assess carotenoid metabolism at concentrations found in the diet. For this reason, it is difficult to obtain concrete structural information for lycopene metabolites using 14C-labeling. Stable isotope labeling provides an alternative low-risk approach, where 13C-labeled carotenoids can be consumed at concentrations found in the diet. This strategy has been used to study β-carotene absorption and conversion to vitamin A [Isotopic labeling has been used as a strategy for investigating carotenoid absorption, metabolism, and excretion in vivo.  rodents ,14 and t rodents . Radiolaitamin A ,17, as witamin A  and phytitamin A  pharmaco13C-label. Plasma samples for this analysis were obtained from a recent study conducted by our group [The objective of this study is to investigate the oxidative metabolism of lycopene in humans using a ur group . We expe13C-labeled aldehyde cleavage products of lycopene in the plasma of subjects from this study. The LC-MS method used for the plasma analysis was, therefore, optimized to detect the various apo-lycopenals expected . This study provided a dose of 10.2 mg 13C-lycopene, which is physiologically relevant. However, masses corresponding to the 13C-labeled apo-lycopenals were not detected in the plasma of the eight subjects at the any of the time points examined. Additionally, native, unlabeled apo-lycopenals were also not detected. Given our group’s previous identification of apo-lycopenals in human plasma, we expected to observe 13C enrichment provided in the tracer dose. This approach was validated with the detection of 13C-lycopene in our plasma samples  was used to search the data and extract mass spectral features with isotopic distributions characteristic of the  samples A. All fe13C-lycopene that was present post-dose, but not at baseline and had the same characteristic reverse isotope distribution as the labeled lycopene , 5-cis-lycopene (18.4 min), and other lycopene cis isomers (14.8–16.8 min), demonstrating structural similarity. From manual review of the ClusterFinder data, we discovered another labeled compound besides lycopene B. With a16.5 min  and eluttrans-lycopene , but quantifiable, on the day of dosing (0–10 h) and increased significantly once subjects resumed their moderate lycopene diet (10–20 mg lycopene/d). At 28 days, the unlabeled lycopene 1,2-epoxide was found to be between 1.63 and 11.18 nmol/L in plasma. We have ruled out the lycopene epoxide being an artifact of sample handling based on the changing ratios of the native (unlabeled) epoxide to native lycopene in plasma over the course of the study. The native lycopene epoxide is present in plasma at 0.089 ± 0.027% the concentration of native circulating lycopene on the day of dosing (0–10 h) when unlabeled lycopene was not consumed. However, the native epoxide is present at 0.52 ± 0.16% and 0.50 ± 0.12% the concentration of native lycopene at 72 h and 28 days, respectively, when subjects returned to a moderate lycopene diet.In this study, we utilized stable isotope technology to detect and characterize the presence of lycopene epoxides in human biosamples. This effort extends the work of Khachik et al. (1997), reporting the presence of lycopene diols in human serum and hypothesizing that epoxidation is an initial step in the oxidative metabolism of lycopene, followed by enzymatic or acid hydrolysis to generate the diol forms ,25. They13C-labeled lycopene dose, we found that one of the peaks in the LC chromatogram originally thought to be a minor cis-isomer of lycopene, was actually the 1,2 epoxide. The 13C-labeled lycopene 1,2-epoxide was present in the dose at approximately 1% the concentration of lycopene. Additionally, the plasma appearance curve of the 13C-labeled lycopene epoxide mimics that of the 13C-labeled lycopene over the first 10 h /methanol (1:1) prior to analysis. All samples were analyzed by HPLC-QTOF-MS the same day they were extracted to minimize potential degradation and oxidation of lycopene and its metabolites. A model 2695 HPLC with a 996 PDA  was coupled to a quadrupole time-of-flight (Q-TOF) Premier hybrid mass spectrometer  via an atmospheric pressure chemical ionization probe operated in negative ion mode. Compounds were separated on a YMC C30 column  (Waters Corp.) with a mobile phase gradient consisting of solvent mixtures A  and B . MS parameters were as follows: corona current, 30 µA; collision energy, 8 eV; cone voltage, 40 V; source block temperature, 110 °C; probe temperature, 600 °C; and desolvation gas flow, 400 L·h−1. All MS experiments were run in Enhanced Duty Cycle mode for increased sensitivity with V-optics enabled (7500 mass resolution). MS data were acquired using MassLynx software version 4.1 (Waters Corp.).The sample preparation and HPLC-QTOF-MS analysis of the plasma were conducted as previously described . Briefly13C-labeled metabolites of lycopene using an untargeted approach. Waters format data files (.raw) were converted to an open data format (.mzXML) using ProteoWizard (version 3.0.10875) [13C isotope patterns within raw mass spectral data. The resulting features were manually assessed and potential 13C-labeled metabolites were evaluated in the full dataset. The HPLC-QTOF-MS plasma data acquired from this study were mined for new 0.10875) . A subseWe have demonstrated that stable isotope labeling is an effective strategy for studying phytochemical metabolites in humans. A lycopene epoxide has been observed in human plasma at biologically relevant concentrations for the first time. As other lipid epoxides have been reported to have biological activity, the lycopene epoxide may contribute to the biological properties associated with lycopene and tomatoes. Follow-up experiments are needed to determine the source of the lycopene epoxide in plasma and to investigate the bioactivity of this oxidation product.


Most of them were small-sized and contained DβH with PACAP, SOM, NPY or GAL. The findings of the present study provide a detailed description of the distribution and chemical coding of the SChG neurons projecting to the skin of the porcine hind leg. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.It is generally known that in the skin sympathetic fibers innervate various dermal structures, including sweat glands, blood vessels, arrectores pilorum muscles and hair follicles. However, there is a lack of data about the distribution and chemical phenotyping of the sympathetic chain ganglia (SChG) neurons projecting to the skin of the pig, a model that is physiologically and anatomically very representative for humans. Thus, the present study was designed to establish the origin of the sympathetic fibers supplying the porcine skin of the hind leg, and the pattern(s) of putative co-incidence of dopamine-β-hydroxylase (DβH) with pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), neuronal nitric oxide synthase, substance P, vasoactive intestinal peptide, neuropeptide Y (NPY), leu5-enkephalin and galanin (GAL) using combined retrograde tracing and double-labeling immunohistochemistry. The Fast Blue-positive neurons were found in the L In the human, autonomic nerve fibers derive from two sources: sympathetic and, rarely, parasympathetic neurons . The symeceptors ,14,15 toeceptors . Severaleceptors ,18,19,201–L4 SChG [6–S2 ganglia [In humans and cats, skin-projecting sympathetic neurons supplying the lower leg/hind paw were distributed in a distinct set of sympathetic chain ganglia (SChG). For instance, in humans such neurons were found in L–L4 SChG , whereas ganglia .Previous studies in the pig showed that the majority of the postganglionic SChG neurons projecting to different organs, i.e., ovary , colon ,25, urin2–L6) and sacral (S1–S2) SChG  SChG .5, while in the sacral region in S1. The total number of FB+ neurons in L2–S2 region was 3231 ± 359, and 73.7 ± 14.0% of them were located in the lumbar segments. Lumbar and sacral skin-projecting neurons were similar in size (782.1 ± 374.6 µm2 vs. 676.8 ± 224.6 µm2). Relative frequency of individual subclasses of porcine skin-projecting SChG neurons, defined by their cross-sectional area is shown in The contralateral SChG did not contain Fast Blue (FB)-labeled neurons. Among lumbar segments the highest number of these cells was observed in LThe majority of FB neurons belonged to the population of small-sized neurons, while large-sized cells was less numerous . RelativWhen the distribution of the FB neurons was compared within four subdomains of the SChG the results were as follows a,b.The small-sized cells were more often located in the cranio-dorsal subdomain (26.6 ± 0.4%) than in the cranio-ventral subdomain (21.4 ± 0.6%) of lumbar SChG. The percentage of these cells was also higher in the cranio-dorsal subdomain of lumbar SChG when compared to the sacral SChG (21.6 ± 2.2%). In the sacral SChG the highest percentage of small-sized neurons was found in the caudo-dorsal subdomain (32.0 ± 1.9%), especially when compared to the lumbar SChG (24.6 ± 0.9%). The distribution of small-sized neurons in the cranio- (22.4 ± 0.6% vs. 19.6 ± 1.3%) and caudo-ventral (26.6 ± 0.9% vs. 26.8 ± 1.3%) subdomains of the lumbar and sacral SChG was similar a.The distribution patterns of the large-sized neurons within the specific subdomains of the lumbar and sacral SChG were quite different b. For exAll details concerning neurochemical phenotypes of skin-projecting neurons within the porcine SChG are demonstrated in The morphology and characteristic features of double-immunolabeled elements are visualized in Double-labeling immunohistochemistry revealed that the vast majority of the small- and large-sized skin-projecting SChG cells displayed immunoreactivity to DβH, indicating the catecholaminergic nature of these neurons. It is worth mentioning that the frequency of the small- and large-sized neurons containing this enzyme was similar in lumbar and sacral SChG .Moreover, these neurons also showed positivity for all other markers tested  but their percentages vary depending on the size and the location alongside the spinal cord.+/DβH+ small-sized cells stained for SOM, NPY and GAL were most numerous in L4, containing PACAP and LENK in L5, while the cells immunorective for FB+/DβH+/VIP+ . It has been found that small-sized neurons were primarily found in the caudo-dorsal subdomain of sacral SChG, whereas the majority of large-sized neurons were found in the cranio-dorsal subdomain of lumbar and sacral SChG. The somatotopic distribution of sympathetic innervating different organs in pig ganglia has been observed by several authors ,36,39,40The present study has disclosed that most of the FB-labeled sympathetic small- and large-sized neurons had a catecholaminergic nature, as revealed by immunoreactivity for DβH. These results correlate well with studies describing the adrenergic character of a substantial part of the SChG neurons in the pig ,27,28,37+ small- and large-sized perikarya also expressed NPY immunoreactivity. NPY is found in cutaneous sympathetic neurons of rodents and in humans [+/DβH+/NPY+ small-sized neurons in the lumbar SChG decrease from the cranial to the caudal direction. It is plausible that these neurons, distributed with different frequencies in the lumbar SChG, can modulate skin functions at different levels. Several FB+/NPY+ small- and large-sized neurons innervating the porcine skin of the hindlimb were non-adrenergic. It is possible that this infrequent population of neurons could act in dependently of NA to induce vasoconstriction, which was previously reported by Stephens et al. [+/NPY+ skin-projecting neurons in pigs remains unexplained and requires further investigation.The present data indicate that a significant amount DβHn humans ,45. Moren humans ,47. Thesn humans ,48,49. Mn humans ,51,52,53s et al.  in human+/DβH+ small- and large-sized SChG neurons was found to stain SOM. SOM has been previously found co-expressed of NA markers in neurons of the porcine SChG supplying the mammary gland [+/DβH+/SOM+ small-sized neurons in the lumbar sympathetic ganglia also decrease from the cranial to the caudal direction.The moderate number of FBry gland , urinaryry gland ,27 and ory gland . A small+/SOM+/DβH+ SChG neurons, since the function of the SOM in connection with NA in skin has also not yet been specified.The SOM-IR nerve fibers have been found in the epidermis, dermis and, rarely, around blood vessels, sweat glands and hair follicles in human ,56. It s6 and S2) were nNOS-IR. The results presented by us may explain the origin of part of the nNOS-IR terminals around microvessels and the glandular duct observed by Ibba-Manneschi et al. [DβH and nNOS were co-expressed in an insignificant percentage of small- and large-sized skin-projecting neurons. The presence of nNOS with DβH had earlier been observed in some SChG neurons projecting to the colon ,25, urini et al.  in humani et al. . Whereasi et al. .+/SP+ nerve fibers have been found in monkey and rat lower lip skin supplying small arteries, veins and arterioles [6 SChG. This finding is similar to that observed by Botti et al. [+ neurons with DβH and alone may partially mediate cutaneous vasodilation through NO-dependent mechanisms [These data revealed that a moderate percentage of adrenergic skin-projecting neurons also contained SP. These co-localizations have been found in the sympathetic neurons projecting to the muscle of the porcine genital tract ,63,64 anterioles . In the i et al. , where ti et al. . Moreovechanisms .+/DβH+ small- and large-sized SChG neurons has been found co-localized with VIP. This kind of co-localization was earlier observed in neurons of porcine SChG [The present results indicate that a part of FBine SChG  projectiine SChG , urinaryine SChG ,27 and eine SChG . Moreoveine SChG ,69. It wine SChG ,71. Baseine SChG ,73.+ small-sized neurons immunoreactive for LENK were non-adrenergic and observed only in S1. It seems possible that, as observed by us, adrenergic and non-adrenergic neurons are a source of very rare nerve fibers in the skin, and may decrease the release of NA and, secondarily, blood pressure, as can be seen from pharmacological studies [This study showed that adrenergic small- and large-sized (only in the lumbar SChG) skin-supplying neurons sporadically co-localized with LENK. This population of neurons had already been found in porcine SChG ,27,59. F studies ,75. The + immunoreactive for PACAP innervating porcine skin of the hindlimb were non-adrenergic. It is possible that skin-projecting SChG neurons might be the source of the DβH+/PACAP+ nerve fibers observed by Steinhoff et al. [+/PACAP+ neurons in the functioning of skin of the hindlimb is still unknown.DβH and PACAP were co-localized in considerable percentages within the skin-projecting SChG small- and large-sized neurons. The co-localization of these substances had already been found in the nerve fibers inside the hypothalamus of rats . Moreovef et al.  close tof et al. ,79. Thus+/DβH+/GAL+ small- and large-sized neurons represent a portion of the retrogradely labeled cells. Additionally, the population of skin-projecting neurons was completely devoid of non-adrenergic GAL-IR cells. The co-existence of these substances was previously observed in SChG supplying the ovary [This study revealed that FBhe ovary . The immhe ovary ,81. It whe ovary . This suhe ovary . Additiohe ovary .In conclusion, the present study provides for the first time a detailed description of both the source of sympathetic nerve fibers innervating the skin of the porcine hindlimb and chemical phenotyping of neurons being the source of these fibers. Moreover, the differences observed by us between the studied ganglia in the percentage of DβH-IR neurons co-localized with NPY, PACAP, VIP, SOM, nNOS, LENK, SP or GAL, suggest that these cells may play a role of modulators of sympathetic control at varying levels of skin function in pigs. Further research should be carried out to elucidate in detail the physiological relevance of each subpopulation of neurons innervating skin of the porcine hindlimb.The experiment was performed on 4 juvenile female crossbred gilts (Pietrain × Duroc), aged 8–12 weeks and weighting 15–20 kg. All animals were housed and treated in accordance with the Principles of Laboratory Animal Care . All experimental procedures were approved by the Local Ethical Commission of the University of Warmia and Mazury in Olsztyn (No. 36/2013). Seven days before surgical operations the animals were transported from a farm to the local animal house, where they were individually housed in stalls, under conditions of natural light and room temperature. All animals were fed with a commercial grain mixture , and they had free access to water. 24 h before surgery feeding was stopped. All efforts were made to minimize the number of animals used and their suffering.12–S4) were collected from all animals studied and then postfixed by immersion in the same fixative for 15 min, washed twice in 0.1 M phosphate buffer  for over three days, and then stored in 18% buffered sucrose solution containing 0.01% sodium azide (pH 7.4) at 4 °C until they sank. Finally, the tissues were frozen and then coronally sectioned at a thickness of 10μm using a cryostat . The sections were stored at −80 °C until further processing.Thirty minutes before the main anesthetic was given, the animals were pretreated with atropine sulfate  and azaperone . All animals were anesthetized with sodium thiobarbital . After induction of the surgical anesthesia, the skin of the left hindlimb was gently shaved, disinfected with 1% water-alcoholic solution of iodinetincture, and then a total volume of 100 μL of 5% aqueous solution of the fluorescent retrograde tracer Fast Blue  was administered in multiple injections in to the external surface of the skin using a Hamilton microsyringe equipped with a 26S gauge needle. Injections were made at equal intervals of one centimeter, and they strictly covered the skin area innervated by cutaneous branches of the femoral and sciatic nerves (100 μL) . Four weThe sections were processed for routine double-immunofluorescence labeling using primary antisera, raised in different species and species-specific secondary antibodies listed in 4–L6, S1 and S2 ganglia. The sections were selected from three different representative regions of the ganglia .Immunohistochemistry involved double-staining, which was applied to sections from L3  and 10% normal goat serum  in 0.01 M PBS.Due to the presence of FB+ neurons in the studied sections, all steps of the procedures described below were performed in darkness. Thus, sections were air-dried at room temperature (RT) for 45 min, rinsed three times in phosphate-buffered saline , and then, in order to block all the unspecific binding sites, incubated for 1h in a humid chamber with a blocking solution containing 1% Triton X-100 , 0.1% bovine serum albumin , 0.05% thimerosal , 0.01% NaNFor the double-labeling immunofluorescence, a mixture of two primary antibodies was used. After the incubation with primary antibodies, sections were rinsed in PBS (3 × 15 min) and incubated for 1 h with a mixture of appropriate FITC-conjugated secondary antisera and biotinylated donkey anti-rabbit antibodies. The latter antibodies were finally visualized by additional incubation of sections with streptavidin-CY3 complex for 1h. Following subsequent rinsing in PBS (3 × 15 min), the sections were coverslipped with carbonate-buffered glycerol (pH 8.6).The sections were viewed under an Olympus BX61 microscope  equipped with epi-fluorescence and appropriate, small-band filter sets for FB, FITC and CY3. Images were acquired by a PC equipped with a CCD camera operated by CellSens Dimension image analyzing software .To avoid a double-counting of labeled neurons, only FB-positive perikary a with a clearly visible nucleus were counted in each fourth section of the particular SChG studied. Moreover, to further unravel the intraganglionic distribution of FB-traced neurons, each of the SChG sections of interest was divided, along their long and short axes, into four subdomains: cranio-dorsal, cranio-ventral, caudo-dorsal and caudo-ventral .The perikarya were divided into two subclasses according to diameter: small  and large  and sacral SChG (S1 and S2).FB+ neurons were counted with in individual lumbar and sacral SChG, as well as within each of the ganglionic subdomains. These counts were expressed as the percentages of small- and large-sized FBU-test. The statistical importance of differences found between ganglia of data was analyzed using one-way ANOVA followed by the Tukey test using GraphPad Prism4 software . Differences between population small- and large-sized neurons innervating skin of the hindlimb were compared using the Mann–Whitney U-test. p < 0.05 was considered to be statistically significant.Data concerning the segmental distribution and the number of skin-projecting sympathetic perikarya in the total population of lumbar and sacral SChG were analyzed using the Mann–Whitney After a careful examination of each injection site, traces of FB were found neither in muscles, nor in the subcutaneous tissues in the vicinity of the tracer deposition places. The specificity of primary or secondary antisera was tested by: (1) preabsorption tests based on incubation with an antibody that had been preabsorbed with synthetic antigen (25 μg of appropriate antigen per 1 mL of corresponding antibody at working dilution); and (2) omission and replacement tests during which the primary or secondary antibody was omitted or replaced by non-immune sera or PBS. Lack of any visible fluorescence indicated the specificity of the labeling.In conclusion, the present study provides for the first time a detailed description of both the source of sympathetic nerve fibers innervating the skin of the porcine hindlimb and chemical phenotyping of neurons being the source of these fibers. Moreover, the differences observed by us between the studied ganglia in the percentage of DβH-IR neurons co-localized with NPY, PACAP, VIP, SOM, nNOS, LENK, SP or GAL suggest that these cells may play a role of modulators of sympathetic control at varying levels of skin function in pigs. Further research should be carried out to elucidate in detail the physiological relevance of each subpopulation of neurons innervating skin of the porcine hindlimb.


Tumor-associated macrophages (TAMs) have also been reported to have prognostic value. However, its clinical significance has not been fully clarified in patients with advanced CRC who present with distant metastases.The immunoscore (IS), an index based on the density of CD3+, CD4+, CD8+, FOXP3+, CD68+, and CD163+ immune cells within CRC tissue procured from three sites–the primary CT, IM, and distant metastasis (DM)–was determined using immunohistochemistry and digital image analyzer (n=196). The IS was obtained by quantifying the densities of CD3+ and CD8+ TILs in the CT and IM. IS-metastatic and IS-macrophage–additional IS models designed in this study–were obtained by adding the score of CD3 and CD8 in DM and the score of CD163 in primary tumors (CT and IM), respectively, to the IS.The density of CD3KRAS mutation and three IS models. However, in the subgroup analysis, IS-metastatic showed a prognostic association regardless of the KRAS mutational status.Higher IS, IS-metastatic, and IS-macrophage values were significantly correlated with better prognosis . Multivariate analysis revealed that only IS-metastatic was an independent prognostic marker (p=0.012). No significant correlation was observed between + and CD8+ TIL densities at DM could be a strong prognostic marker for advanced CRC.IS is a reproducible method for predicting the survival of patients with advanced CRC. Additionally, an IS including the CD3 Tumor-infiltrating immune cells can influence tumor progression and metastasis. While one of their functions is recognition and elimination of tumor cells , they ha+ and CD8+ lymphocytes in the tumor center (CT) and invasive margin (IM) [Recently, several studies have demonstrated that TILs have high prognostic utility. Galon et al. introduced the ‘immunoscore (IS)’, a value based on the density of CD3gin (IM) –19. Moregin (IM) , 21. Howgin (IM) , 22.Tumor-associated macrophages (TAMs) are another component of tumor-infiltrating immune cells. Macrophages are derived from monocytes and exhibit two polarization states in response to different microenvironmental signals–M1 and M2 –25. M1 mThe aim of this study was to confirm the prognostic value of the IS in patients with advanced CRC. The characteristics of tumor infiltrating immune cells was also determined. Additionally the heterogeneity in the target sites of tumor-infiltrating immune cells in patients with advanced CRC was evaluated.+ lymphocyte was the highest in the IM ; 389.15, 246.95–649.42) than any other site . The density of CD8+ lymphocytes was lower in the CT  than the IM  and the DM . The pixel count per area (pixels/mm2) of CD68+ macrophages was the highest in the DM . Similarly, CD163-positive macrophages were more frequently infiltrating in the DM  than any other site. All tumor-infiltrating immune cells except FOXP3+ lymphocytes presented a heterogeneous density according to tumor location . A low density of CD4+ lymphocytes in the CT and IM was also related to a poor outcome . In contrast, there was an association between CD8+ and FOXP3+ lymphocytes in the DM and patient worse outcome . The patients presenting with a high density of CD68+ and CD163+ macrophages in the CT of their primary tumor had significantly worse outcomes. Additional data on the median survival time and comparisons between patient groups according to immune cell markers and respective tumor location are listed in We divided the patients into low and high groups by the predetermined cut-off values of the continuous variables using maximal chi square method according to each immune cell marker in each tumor location. The Kaplan-Meier method revealed that a low density of CD3Through the assembled density data, we evaluated the patients with the IS system, which gives a score depending on the total number of high densities marked (from IS0 to IS4). Owing to loss of tissue microarray (TMA) core tissue, IS results were available for only 193 of 196 patients. According to the IS, 49.7% (96/193) was recorded as a low IS  and 50.3% (96/193) was a high IS (IS3: 43.0% and IS4: 7.3%).In the present study, we used two additional scoring models that incorporate the results of macrophage infiltration into the primary tumor  and lymphocyte infiltrates into distant metastases . IS-ma and IS-M results were available in 193 and 188 patients, respectively. Seventy five (38.9%) patients were low IS-ma  and 118 (61.1%) patients presented with a high IS-ma . Of 188 patients, 53.2% (100/188) had low IS-M  and 46.8% (88/100) had high IS-M .When the IS was compared to the patient's clinicopathologic features, higher pT stage (p = 0.001) and the presence of perineural invasion (p = 0.008) were significantly correlated with lower IS Table . Lower IThe Kaplan-Meier analysis revealed that all three IS models had a prognostic association. Higher scores were significantly correlated with improved survival  Figure . By univMultivariate COX regression analysis revealed that of all three IS models, only the IS-M model was an independent prognostic factor (p = 0.012) Table . Older aKRAS and 106 (54.4%) had mutated KRAS. Among the tumors with mutated KRAS, mutations in codon 12 or 13 were identified in 99 (93.4%). Additionally, mutations in BRAF (V600E) were identified in 7 patients (3.6%), and those in PIK3CA were identified in 25 patients (13.1%). The two most common PIK3CA mutations were found in exon 9  and exon 20 .Of the 196 cases examined, 89 (45.6%) had wild-type KRAS or PIK3CA mutations. In BRAF-positive patients, the density of CD4+ and FOXP3+ T cells was significantly low  in the CT, whereas FOXP3+ T cell density was significantly high (p < 0.001) in the IM. The density of CD163+ macrophages in the IM was significantly high in patients with KRAS mutation (p = 0.038).There was no difference in the T cell densities of tumors with KRAS, PIK3CA, and BRAF mutations had no significant prognostic association. In subgroup analysis, IS-M and IS-ma showed significant prognostic association regardless of the KRAS mutational status. The IS showed a prognostic association in KRAS mutation-negative group . Four of these 7 patients died during the follow-up period; all 4 had low IS, IS-M, and IS-ma.Kaplan-Meier survival analysis revealed that p Figure . IS-M anWe regrouped the patients into low IS and high IS groups by the median values of the continuous data of immune infiltrates. IS, IS-M, and IS-ma were recalculated by summing the scores assessed by median cut-offs. Of 196 patients, 129 (65.8%) had low IS  and 67 (34.2%) had high IS (IS3: 20.9% and IS4: 13.3%). One hundred and twenty-five (64.7%) patients had low IS-ma , and 68 (35.3%) patients presented with high IS-ma . Of 188 patients, 117 (62.2%) had low IS-M , and 71 (37.8%) had high IS-M .When the IS was compared to the patient's clinicopathologic features, lower IS was associated with synchronous metastasis (p = 0.035). Lower IS-M was also correlated with aggressive clinicopathological features, including higher pT stage (p = 0.009), pN stage (p = 0.012), synchronous metastasis (p = 0.010), and perineural invasion (p = 0.016). There was no significant association between IS-ma and clinicopathologic features. The Kaplan–Meier survival analysis revealed that a high score of IS-M was significantly associated with good prognosis (p = 0.008), although IS and IS-ma models had no significant association .+ and CD8+ lymphocytes was higher in the IM than in the CT. The infiltration of CD3+, CD8+, and CD163+ immune cells was significantly different between the CT and the IM as well as between the CT and DM. Although several studies have reported the density of tumor-infiltrating immune cells of CRC patients according to the tumor sites, these reports included only two or three subsets of tumor-infiltrating immune cells or did not consider the density of tumor-infiltrating immune cells in the DM [The present study demonstrated that TILs and TAMs show significant heterogeneity in their tumor infiltration site. The density of CD3n the DM –33. This+, CD8+ or CD45RO+ lymphocytes are associated with less aggressive clinicopathological features and a better prognosis [In several previous studies, the protective role of T cell subsets on tumor progression has been consistently reported –36. Mostrognosis , 36. In Our results demonstrated that the IS-M model, which includes the score of two lymphocytic markers in the DM, is superior to IS or IS-ma. The conventional model designed by Galon et al. covers lymphocytic infiltrates in only the primary tumor. However, in the present study, we confirmed that tumor-infiltrating immune cells have not only heterogeneity of quantity but also distinct clinical significance in relation to the tumor location. Therefore, the immune infiltrates in metastatic lesions as well as in the primary tumor should be assessed to validate the patient's systemic immune reaction on whole tumors. This is supported our by results showing that the IS-M model was the only independent prognostic marker among the IS models in multivariate analysis.KRAS or PIK3CA mutational status. Hence, immune contexture, including immune cell density in primary and metastatic tumors, could be a reliable prognostic marker in CRC, regardless of patients' mutational status.In a recent report, Lea et al. described the limitations of the current TNM staging system in predicting the outcome of patients with CRC . They suth percentile, median, and 75th percentile could be candidates for alternative cut-off values. We calculated three IS systems using the median cut-off, and the results of survival analysis showed a similar tendency; the patients with a higher IS-M score had significantly better outcome. However, both the cut-off values calculated by the maximal-chi square method and the median values would differ according to several factors, including cohort characteristics, quality of the sample, selected area of examination, antibodies to be used, and cell counting algorithms. Thus, to set a reliable cut-off value of the density of immune infiltrates, a multicenter prospective study for the standardization of the detailed methodology is needed.However, it seems that there are some challenges in applying the IS system as a prognostication factor. First, the determination of an optimal cut-off value is difficult. Galon et al. illustrated that a predetermined cut-off value should be used to score high versus low values for each marker in each location. The previous study by Galon et al., as well our study, used a maximal-chi square method to set optimal cut-off values. However, other values such as the 25Another challenge is the selection of optimal area for density analysis. Since the density of immune infiltrates is highly heterogeneous, selection of the analyzed area could affect the results. According to Galon et al., who first suggested the IS system, the combined analysis of immune infiltrates in the CT and IM could improve the prediction of patient survival . After tIn summary, we demonstrated the regional heterogeneity of tumor-infiltrating immune cells according to the tumor location in our large cohort of advanced CRC patients with synchronous and metachronous distant metastasis. Also, the amount of immune infiltrates was also heterogeneous in relation to the metastatic organ examined. Higher infiltrates of TIL and lower infiltrates of TAM correlated with longer survival. The three IS models, IS, IS-ma, and IS-M also had prognostic significance in univariate analysis. Among the three IS methods, the IS-M model that includes TILs in the DM was an independent prognostic marker. Our results suggest that immune infiltration in the DM should be evaluated to assess the IS system for advanced CRC patients with distant metastases.A total of 196 advanced CRC patients who presented with synchronous or metachronous metastases were enrolled in this study. They underwent surgical treatment for primary and metastatic disease at Seoul National University Bundang Hospital  between 2003 and 2009. Of the 196 patients, none had received preoperative systemic therapy or radiation treatment. The patient's clinical and pathological data were obtained through medical charts and pathology reports. The patient outcomes and their survival times were collected. The patients lost to follow-up or dead from causes other than CRC were assumed as censored. The follow-up period ranged from 0.8 to 104.6 months .All patients with synchronous metastasis underwent adjuvant chemotherapy after the surgical resection of primary and metastatic tumors. Of the 62 patients with metachronous metastasis, 56 underwent adjuvant chemotherapy, and presented with metastatic lesions during their follow-up period. Six patients with metachronous metastasis treated with curative resection of primary cancer received no adjuvant chemotherapy after the surgical resection. Since the metastatic lesion was observed during their follow-up period, they were treated with metastasectomy and chemotherapy.Formalin-fixed paraffin-embedded tissues from the CRCs were collected. The representative core tissues (2 mm in diameter) were used. The obtained tumor tissue included the area of CT and IM of the primary tumor as well as its related DM. Each core tissue was rearranged into tissue array blocks using a trephine apparatus  .The presence of tumor-infiltrating immune cells was confirmed by immunohistochemistry using antibodies for CD3 , CD4 , CD8 , FOXP3 , CD68 , and CD163 . Immunostaining for CD3, CD8, and FOXP3 was performed using a Bond polymer kit (Leica Microsystems) and Leica BOND-MAX autostainer (Leica Microsystems). CD4, CD68, and CD163 expression was detected immunohistochemically on a Ventana Bench mark XT autostainer (Ventana) with the OPTIVIEW universal DAB kit (Ventana).® CS instrument  at 20 x magnifications. Each immunomarker-positive tumor-infiltrating immune cells quantified by computerized image analysis system, ImageScope™ (Aperio Technologies)  Figure. CD3+, CThe patients were divided into two groups by the density of each tumor-infiltrating immune cell according to each tumor location (high vs low). To set the best cut-off values, the maximal chi-square method was used related to the patient's overall survival , 38. In + and CD8+ TILs in metastatic tumors. It is a summation of the score of CD3+ and CD8+ TILs in the CT, IM, and DM.The IS is defined as a quantification system based on the combination of two markers (CD3 and CD8) in two regions , 19. A h+ TAMs in the primary tumor (CT and IM) to the IS. Thus, IS-ma includes the score of CD3+, CD8+, and CD163+ immune infiltrates in CT and IM. Our data showed that TAMs had an opposite prognostic correlation compared to that of TILs; a high density of TILs was recorded as score 1, but a high density of TAMs was recorded as score 0 to ensure the consistency of the scoring system. The schematic definitions of each of the three IS models are described in Figure Another score model, IS-ma, is calculated by adding the score of the density of CD163KRAS, 4 ng/μL; BRAF, 5 ng/μL; and PIK3CA, 2 ng/μL). Amplification and detection were performed using an Automated Cobas X480 analyzer. The real-time PCR assay was performed to detect the mutation in codons 12, 13, and 61 of KRAS; the V600E BRAF mutation; and the mutation in exons 1, 4, 7, 9, and 20 of PIK3CA.Hematoxylin-Eosin (HE)-stained slides of CRC tissues were reviewed by a pathologist (H.S.L). Tumor areas were identified and microscopically dissected to sections with an area of more than 1 × 1 cm and comprising more than 60% tumor cells. One or two 8-μm-thick formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were de-paraffinized in xylene for 5 min at room temperature (RT), dehydrated in absolute alcohol for 5 min at RT, and air dried completely for 10 min. DNA was isolated using the Cobas DNA Sample Preparation Kit  according to manufacturer's instructions, and the same preparation protocol was followed for all Cobas mutation kits used in this study. The concentration of the isolated DNA was measured using a NanoDrop UV spectrophotometer , and the DNA was diluted with DNA Specimen Diluent from the Cobas 4800 Mutation Test kit (Roche) to the optimal concentration for each gene (http://cran.r-project.org/). The Kaplan-Meier method was used to examine survival outcomes and the significance of the differences between groups was compared using the log-rank test. A univariate and multivariate regression analysis was performed using Cox proportional hazards models to determine hazard ratios (HRs). P values of less than 0.05 were considered statistically significant. All statistical analysis, except for the maximal chi square test, was performed using IBM SPSS statistics 20 .To compare each non-continuous variable, a Wilcoxon/Mann-Whitney test or Kruskal-Wallis analysis was used. To establish the optimal cut-offs of continuous variables, the maximal chi-squared method was performed using the R program (


This study is aimed to compare the diagnostic yield, complications and influencing factors between Radial endobroncheal ultrasonography guided bronchoscopy(R-EBUS) and CT-guided needle biopsy (CT-PNB), for evaluation of solitary pulmonary nodules(SPNs).160 cases of consecutive patients with SPNs were enrolled and divided into R-EBUS and CT-PNB groups randomly. The diagnostic yield, complications and influencing factors between the two groups were evaluated.P = 0.006, P = 0.002). In R-EBUS group, the factors affecting diagnostic yield were size (P = 0.027), the distance between SPNs and pleura (P = 0.031) and the location of the probe to lesions (P = 0.009). In CT-PNB group, the distance from the lesions to pleura was correlated with the incidence of pneumothorax (P = 0.001) and pulmonary haemorrhage (P = 0.042). The location of SPNs were adjacent to great vessels was another influencing factor for pulmonary haemorrhage (P = 0.042).Sensitivity of R-EBUS for malignancy was 73.7% (42/57) and for benign, was 43.5% (10/23), overall diagnostic accuracy was 65% (52/80). In CT-PNB group, overall diagnostic accuracy was 85% (68/80), sensitivity for malignancy was 87.9% (51/58), and for benign was 81.0% (17/21), respectively. Both overall diagnostic yield and incidence of complications in CT-PNB group were higher than those in R-EBUS group (Both R-EBUS and CT-PNB are valuable tools for diagnosis. SPNs located in medial 1/2 of lung field, or were adjacent to great vessels may be fit for R-EBUS. Those SPNs located in lateral 1/2 of lung field, near to pleura or with less vessels around may be more suitable for CT-PNB. Single pulmonary nodules (SPNs) is defined as single, isolated lesion of circular or ovoid shape, with the diameter of ≤ 30 mm, which is located within the lung parenchyma, surrounded entirely by gas-containing lung tissue and not accompanied wiht hilar enlargement, pleural effusion or lung atelectasis . With thP > 0.05). The average diameter of SPN (mean ± SD) was (2.17 ± 0.31) cm in the R-EBUS group and (2.09 ± 0.30) cm, in the CT-PNB group. (added to Table Table D) was 2.7 ± 0.31 As shown in the Table Three factors affecting diagnostic yield in the R-EBUS group were listed as follows Table : (a) sizP = 0.027).Further analysis was performed to indicating that in 3 subgroups with different SPNs size, those SPNs with the diameter between > 8 mm and ≤ 10 mm, were not diagnosed. Those SPN with diameter > 20 mm and ≤ 30 mm had higher diagnostic yield than that those with their diameter > 10 mm and ≤ 20 mm cases (P = 0.031).Based on difference in distances from the lesion to pleura, we divided all patients with SPNs into three subgroups . From the data in Table P = 0.009).According to the relationship between the inserted probe and the SPNs’ location, the ultrasonic images were categorized into three patterns: (a) invisible: abnormal echogenicity was not completely detected; (b) within: entire circumference of abnormal echogenicity was detected; (c) adjacent to: partial circumference of abnormal echogenicity was detected; . However, these two factors were not observed to influence the diagnostic accuracy in CT-PNB group .Further logistic analysis results in Table Comparison of incidence of compliactions (pneumothorax and pulmonary hemorrhage) and influencing factors between the R-EBUS and CT-PNB groups.P = 0.001).As listed in Table P = 0.900, and P = 0.842, respectively)  Table .P = 0.001). Furthermore, the distance between the lesion and pleura was also another influencing factor for pulmonary hemorrhage (P = 0.042). The Table P = 0.009).Further analysis showed in CT-PNB, no significant difference of incidence of pneumothorax among three subgroups according to size of SPNs. Base on the different distance from the SPN lesion to pleura, we found no pneumothorax occured in the subgroup in which the distance from SPN to pleura was ≤ 20 mm . However, a significant difference was found between the rest two subgroups in which distance from lesion to pleura ranged was 20 mm to 40 mm or > 40 mm (P > 0.05)  Table .From Table Our data demonstrated that the diagnostic yield in CT-PNB group were higher than that in R-EUBS group. The reason is that the guidence of CT scanning could make clear whether puncture needle had entered into the SPN lesion before biopsy, while R-EBUS’s guidence can hardly perform such real-time supervision. Therefore, the diagnostic yield with R-EBUS’s guidence was lower than with CT-PNB but still maintained the diagnostic accuracy of 65% .As mentioned in our results, size of SPN was important factors affecting the diagnostic yield in R-EBUS group, which has been described by many other reports –16, 21. Data in Table Many studies have reported the position of probe could affect the diagnostic yield of R-EBUS , 16. TheThe overall incidence of complications as pneumothorax and hemorrhage, in R-EBUS group, were 1.25% 1/80) and 5% (4/80). In CT-PNB group, the rate of pneumothorax was 17.5% (14/80), of hemorrhage, was 7.5% (6/80). Pneumothorax is one of the most common complications in lung puncture [0 and 5% Data in our study showed that in CT-PNB group, the longer the distance from the SPN to the pleura was, the higher the incidence of pneumothorax could be. This phenomenon could be explained by the fact the longer the distance from lesion to pleura was, the more pulmonary alveolus were possibly damaged by the needles passing through , 14, 24.Our data showed the incidence of another common complication, pulmonary hemorrhage between the R-EBUS and CT-PNB group were similar. Subgroup analysis listed in CT-PNB group demonstrated that different distances from the SPNs to the pleura had different incidences of pulmonary hemorrhage. However, no similar changes were found in R-EBUS group. This phenomenon may be explained that the longer the distance from the SPN to pleura was, the more pulmonary tissue and vessel may be hurted by puncture needle. The R-EBUS was performed in human natural bronchus lumen so that such injury could be avioded. Among 6 cases with pulmonary haemorrhage in CT-PNB group, 5 cases were found their SPN lesions were near the great vessels. It indicated the richer the blood supply to SPN was, the more easily the pulmonary hemorrhage took place, which was also supported by some recent literature . Our furIn conclusion, both R-EBUS and CT-PNB are valuable tools for diagnosis of SPNs. Those SPNs location within the branches, near to the hilum or great blood vessels in chest, may be suittable for R-EBUS. Those SPNs located in peripheral lung field, near to pleura or lack of great vessels around may be fit for CT-PNB. It is important for clinicians to select strictly appropriate patients with SPNs for achieving high diagnostic yield and low incidence of complications.There are several limitations in our study. First, the study was performed by a single medical institution. Second, different skills and proficiency levels of the three endoscopists participated in this study could produce bias. Third, the size of SPNs and the distance between the lesion and pleura were measured in line, which possibly cause bias compared with the measurement in three-dimensional forms. Strictly controlled, randomized and multi-center clinical research trails are still needed in the future .We have adhered to Optimization scheme of randomized trials of non-pharmacologic Treatment as the CONSORT guidelines showed , 25, 26.Sample size in our study was estimated by algorithm via Inequality Tests for Two Proportions in the menu of PASS 8.0 software. with power 1-β = 0.90, Inspection level α = 0.05. According to the preset parameters, the PASS 8.0 software calculates that the two groups need 80 cases respectively. Therefore, this study expects two groups to have 80 cases respectively, which can ensure the accuracy and scientific results of the study.The actual procedure are as follows: a computer engineer first generated a random number table  with the SAS 9.4 software, then another information engineer gave 1–160 Figures to each patient in the order of time when they were admitted to hospital. The recruited patients were divided into two groups (A:R-EBUS group and B:CT-PNB group) according to the random number table.Bronchoscopies and percutaneous lung puncture were conducted at the Endoscopic Center of Nanjing Chest Hospital.The examination was performed by three experienced endoscopists in turn.215 patients with SPN detected by spiral CT were consecutively enrolled from June 2014 to June 2016. According to the inclusion/exclusion criteria, finally 160 eligible patients were randomly allocated into either R-EBUS or CT-PNB group by random number selection. Before the operation all patients received lung and heart function, electrocardiogram and coagulation testing. Assessment about characteristics of SPN images in high resolution CT were recorded by two experienced radiologists. After the operation, all patients received a 24-hour medical observation for handle complications if necessary. Patients who were non-diagostic by R-EBUS or CT-PNB received VATS operation or antituberculous or antifungal therapy. All patients were followed up for at least 1 year . The locThe characteristic of lung lesions accorded with the definition of SPN. Clinical and imaging data were visible. Patients who agree to sign informed consent.Severe emphysema, multiple or single bullae in lung parenchyma near to SPNs. cardio or pulmonary function insufficiency. hemorrhagic diseases or coagulation disorders. the diameter of SPN < 8 mm whether anywhere in lung fields. The patient underwent mental disorders or those can not cooperate the examination.All bronchial were performed via nose route under local anesthesia and moderate sedation. An video bronchoscope  equiped with a 20-MHz radial EBUS probe  advanced along bronchus then the radial probe were inserted forward until the ultrasonic image indicating the target lesion clear. The radial ultrasound probe position were divided into three patterns as previously reported –16, 21. Patients lay on inpection table in supine, prone, side or other position for finding out the shortest distance from the SPN lesion to the body chest surface. CT images were controlled to scan by a slice thickness of 5 mm to determine the puncture point, depth and direction. After marking on body surface, sterilization and local anesthesia, an introducer needle  was inserted into the SPN lesion under CT guide. If CT image comfirmed the tip of the puncture needle had entered the lesion, the patient was advised to hold his/her breath, then a cutting needle was stretched into the lesion via the introducter trocar. Repeatedly sampling was performed till appropriate specimens had been obtained. The procedure may be stopped by obvious cough, chest pain or any other discomfort symptom. After needle removal, CT scanning were carried out to detect if any complication such as pneumothorax and haemorrhage occur, for necessary intervene.(a) Pathological results of specimen were determined malignancy or identified pathogens as tubercle bacillus, fungal spore or mycelium etcetera.(b) Histopathological showed granulomatous/inflammatory and the size of SPN remarkably reduced or disappear in CT image after experimental standardized antituberculosis/antibiotic therapy.(a) Histopathological considered inflammatory but no shrink in size of leison after sufficient antibiotic therapy and finally were proved to be malignant by surgical resection.(b) Histopathological findings showed abnormal shape cell or suspicious malignant cell but later determined to be a benign lesion by surgical resection.t-test, and dichotomous variables were analysed with the chi-squared or the difference in frequency or Fisher’s exact test when necessary. Logistic analysis were used to calculated relevance. A two-tailed P-value of less than 0.05 was considered significant.Data analyses were performed using SPSS statistical software . Continuous variables were analysed using Student’s


Intracellular uptake and cytotoxicity were assessed in two cell lines  by employing inductively-coupled plasma-mass spectrometry (ICP-MS) analysis and a cell viability assay based on 3--2,5-diphenyltetrazolium bromide (MTT), respectively. We found that the collagen-coated gold NPs exhibit lower cytotoxicity, but higher uptake levels than PMA-coated gold NPs. These results demonstrate that the surface coating of Au NPs plays a decisive role in their biocompatibility.We studied the physico-chemical properties , cellular internalization and toxicity of gold nanoparticles (NPs) stabilized with the most abundant mammalian protein, collagen. The properties of these gold NPs were compared to the same sized gold NPs coated with synthetic poly(isobutylene- The development of new biocompatible nano-materials is an exponentially growing field of research. For example, the number of potential biomedical applications for gold nanoparticles (Au NPs) has expanded impressively in recent years. A vast part of this research is dedicated to the synthesis of Au NPs that could be employed in various medical fields , such asalt-maleic anhydride) (PMA) [In this study, we compared two Au NP systems differing in surface coating in terms of their interaction with cells and culture media. One system was coated with a synthetic polymer, dodecylamine-modified poly(isobutylene-e) (PMA) ,9, and te) (PMA) ,11.Collagen has both a reducing and a stabilizing role in Au NP formation. A stock solution of gold salt was prepared by dissolving 1 g hydrogen tetrachloroaurate (III) hydrate  in 50 mL ultrapure water. A solution of collagen was prepared by mixing 10 mL ultrapure water with 0.02 g of collagen from bovine Achilles tendon  in the presence of 500 µL hydrochloric acid 37% (Sigma-Aldrich). One-point-five milliliters of collagen solution (0.02 g collagen in 10 mL water) was mixed with 0.5 mL ethanol. Subsequently, 90 mL of ultrapure water were added to the collagen-ethanol solution, which was then mixed with 1 mL of gold salt solution. The mixture was heated and stirred until boiling. When it started to boil, the solution was neutralized by quickly adding 2 mL of 1% sodium hydroxide, and the heating was turned off. Instantly, the solution turned into a wine-red color, and its pH was 7. Sodium hydroxide (Sigma-Aldrich), ethanol and sodium chloride  were of analytical grade. All solutions were prepared in ultrapure water with a resistance higher than 18 MΩ . The protocol is given in full detail in the Supporting Information. The Raman spectrum of collagen-coated Au NPs is presented in 7 M−1·cm−1) [Collagen-coated Au NPs were filtered three times and then suspended in bicarbonate buffer pH = 8.6. Their concentration was calculated from the UV-VIS absorption spectrum (molar extinction coefficient ε = 2.03 × 10−1·cm−1) . One mil4 was transferred to toluene to form an ionic pair with TOAB (4.5 eq.), which also acts as a stabilizing agent. In the organic phase, sodium borohydride  was added to reduce Au3+ to Au0, leading to the formation of colloidal Au NPs. The NPs were then washed to remove the excess of ions with HCl, NaOH and, finally, Milli-Q water. Each time, the added aqueous phase was discarded. Ostwald ripening, facilitating the formation of a monodisperse suspension, occurred during an overnight incubation. 1-dodecanethiol was added to replace the surfactant ligands on the particle, rendering the NPs more stable. NPs were further purified with methanol and finally re-dispersed in chloroform. It must be noted that upon ligand exchange of TOAB to 1-dodecanethiol, part of the original TOAB molecules may be left on the Au surface. Ligand exchange processes are known to be not always 100% complete [Mw = 6000 g mol−1), as described previously [7 M−1·cm−1, core diameter dc = 4.4 nm, thickness of surfactant layer l = 1.2 nm, leading to the effective diameter deff = 6.8 nm, number of added polymer monomer units per effective NP surface area RP/area=200 nm−2. Seventy five percent of the anhydride rings of the polymer were linked with dodecylamine. Optionally, the fluorophore Dy647 was incorporated into the polymer, as described in previous reports [Hydrogen tetrachloroaurate (III) hydrate (Alfa Aesar), sodium borohydride (Sigma-Aldrich) and tetraoctylammonium bromide  were used to synthesize Au NPs (4–5 nm in diameter) according to previously published protocols ,13. Briecomplete . A detaieviously ,9,15,16. reports . Hereby,cf.cf.dc of the NPs were calculated from the TEM micrographs using the ImageJ 1.45s software from Wayne Rasband, NIH, USA. The hydrodynamic diameter, dh, and the zeta potential, ζ, of Au NPs were assessed with a Malvern Zeta-sizer , based on dynamic light scattering (DLS) and laser Doppler anemometry (LDA), respectively.Both types of NPs were filtered and then purified by gel electrophoresis cf.. Two per2. Cells were seeded on 96-well plates at a density 5000 cells/well 24 h before experiments.Cervical carcinoma (HeLa) and adenocarcinoma alveolar basal epithelial (A549) cells were cultured in Dulbecco’s Modified Eagle’s culture medium  supplemented with 2 mM glutamine (Sigma-Aldrich), 10% FBS  and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). The cells were grown at 37 °C in a humidified atmosphere containing 5% CO3 and after 5 min, added to each sample. This was followed by the addition of 2.7 mL of 2% HCl. This treatment ensured sample decomposition to near atom size and allowed for more accurate measurements [A549 and HeLa cells were plated in 96-well plates at a density of 5000 cells/well 24 h prior to the experiments. The cells were incubated with different concentrations of Au NPs for 4 h. Au NPs were incubated with the cells in media with or without serum. The supernatants were collected and stored for further analysis at −20 °C. The cells were cultured for an additional 20 h. Then, the medium was removed, and the cells were lysed with a lysis buffer . After 30 min, the samples were collected and stored at −20 °C. Prior to ICP-MS measurements, 150 µL of 37% HCl were mixed with 50 µL of 65% HNOurements .Toxicity induced by gold NPs was evaluated with the MTT assay . The asscf.dc = 7.4 nm, i.e., excluding the contribution of the collagen coating to the size, which does not provide contrast in TEM [We developed a synthesis procedure for Au NPs in which collagen acts both as a reducing and as a stabilizing agent. The one-step synthesis of protein-coated Au NPs prevents the use of reducing agents and stabilizers of a chemical nature. Thus, no surface modification step is required, and the synthesized Au NPs are exclusively covered by a protein layer, which in principle could be an advantage in terms of biocompatibility. In contrast to other techniques, in which Au NPs are generated free of ligands by laser ablation and then covered by adsorbed proteins , in the dh ≈ 13.5 ± 0.5 nm and ζ = −58.4 ± 2.5 mV, respectively , as well as the zeta potentials (ζ) are similar for both types of NPs, which suggest similar physico-chemical properties of collagen and PMA Au NPs in water. The high negative zeta potential values indicate a good colloidal stability in water. However, the surface coating of both types of NPs is quite different, one being a protein and the other a synthetic polymer.The hydrodynamic diameter obtained by DLS from the number distribution in water and the zeta-potential were = 4.4 nm D, a hydrin water , respectcf.i.e., supplemented DMEM). Quite differently, collagen-coated Au NPs were stable in medium with serum, but were prone to form aggregates in medium without serum. Similar results were found for bovine serum albumin-coated Au NPs when exposed to comparable media [The colloidal properties of NPs, however, are strongly dependent on the medium in which they are dispersed. The presence of salt may screen the charge of the NPs, and proteins may be adsorbed to the NPs surface . The colle media ,26. Our We compared the uptake of both types of Au NPs by HeLa and A549 cells. First, the uptake of NPs was visualized with fluorescence microscopy. For this purpose, cells were incubated with Au NPs, which were labelled in their collagen or PMA shell with Dy647. In cf.cf.c than the PMS-coated NPs, cells were exposed to more Au atoms in the case of collagen-coated Au NPs. One Au NP with a core diameter dc comprises NAu = (4π/3)·(dc/2)3·ρAu/MAu Au atoms, using the density of bulk gold ρAu =19.32 g/cm−3 and the molar mass of one Au atom MAu = 196.96 g/mol [NAu(collagen NP)/NAu(PMA NP) = (dc(collagen)/dc(PMA NP))3 = (7.4 nm/4.4 nm)3 ≈ 4.75-times more Au atoms than one PMA-coated NP. Thus, if cells are incubated with the same number of NPs added, in the case of collagen-coated NPs, cells are incubated with a 4.75-times higher amount of elemental Au than upon incubation with PMA-coated NPs. Even considering this fact, that at equal incubation concentrations of collagen- and PMA-coated Au NPs, cells were incubated with a 4.75-times higher concentration of elemental Au in the case of collagen-coated NPs, the difference in internalized elemental Au is much larger in the case of collagen-coated NPs. Furthermore, in terms of the number of NPs, the collagen-coated NPs were internalized to a much higher extent.For quantitative uptake studies, the cells were incubated with the Au NPs in the absence of serum, as well as in serum-supplemented media, and NP uptake was quantified as the amount of internalized Au atoms via ICP-MS; 96 g/mol . Thus, oversus the PMA-coated NPs was caused by agglomeration of the collagen-coated Au-NPs, causing them to sediment on top of the cells and, thus, to be incorporated faster. However, increased uptake was also observed under serum-containing conditions, in which the collagen-coated Au NPs did not agglomerate to an appreciable extent. Thus, it appears that collagen versus PMA surface coating may have a direct effect on the extent to which NPs are taken up, apart from the effect of agglomeration.In the case of serum-free incubation conditions, it might be argued that the increase in NP uptake of the collagen- The potential of Au NPs towards future medical applications can be properly evaluated only if possible toxic effects on cells are taken into consideration. Therefore, we assessed the impact of collagen- and PMA-coated Au NPs on the viability of A549 and HeLa cells. The NPs were incubated with the cells in the presence of serum. The cells were exposed to a range of Au NP concentrations between 0.2 nM and 200 nM. As shown in in vitro as in vivo [In this work, we characterized spherical collagen-coated Au NPs with a mean core diameter of 7 nm. These NPs were obtained by following a straightforward one-step synthesis method. Collagen acted as a reducing as well as a stabilizing agent. Internalization and toxicity of collagen-coated Au NPs were tested in A549 and HeLa cells. In contrast to common synthesis protocols, in which Au NPs are first prepared with different, often synthetic, ligands, and then protein coating is achieved by ligand exchange and/or adsorption, here, the Au NPs are directly grown with a native collagen shell. This prevents the presence of any residual synthetic ligand on their surface. The results obtained for the collagen-coated NPs were compared to the data for Au NPs in the same size range, but coated with a synthetic polymer (PMA). NPs stabilized with collagen were taken up by both cell lines more efficiently than those stabilized with PMA, both in the presence and absence of serum. Concentration-dependent toxicity studies further revealed that at short exposure times, collagen-coated Au NPs were less toxic to the cells than PMA-coated Au NPs. Importantly, the collagen-coated NPs, which are internalized to a high degree, exhibit lower toxicity. We speculate that the lack of any synthetic ligands on the surface of collagen-coated NP may be beneficial, whereas in the case of PMA-coated Au NPs, part of the toxicity may originate from residual TOAB fragments due to incomplete ligand exchange. It is also important to note that the coating of the Au NP surface will not remain stable after the NPs have been internalized by cells. This has been demonstrated for both particle types. For Au NPs coated with proteins, it has been shown that a fraction of the proteins may be enzymatically degraded or exchanged with other proteins present in different endosomal compartments . Further in vivo . Still,


Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli. This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health.The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in  Escherichia coli, in addition to its pleiotropic effects, this protein plays a determinant role in bacterial motility and biofilm formation regulation. Similarly, the bacterial second messenger c-di-GMP is a molecule with high importance in coordinating the switch between planktonic and sessile life in bacteria. Here we have unravelled the importance of accurate regulation of cross-talk between BolA and c-di-GMP for a proper response in the regulation of these bacterial lifestyles. This finding underlines the complexity of bacterial cell regulation, revealing the existence of one additional tool for fine-tuning such important cellular molecular mechanisms. The relationship between BolA and c-di-GMP gives new perspectives regarding biofilm formation and opens the possibility to extend our studies to other organisms with relevance for human health.Bacterial cells have evolved several mechanisms to cope with environmental stresses. BolA-like proteins are widely conserved from prokaryotes to eukaryotes, and in  Escherichia coli (E. coli) bolA gene is induced at the onset of stationary phase and in response to several stresses, leading to substantial changes in the cell  polymerase I (PAPI)  .BolA and its homologues constitute a protein family that is widely conserved across prokaryotes and eukaryotes . FunctioOne of the most extensively studied factors involved in the transition from the motile state to the non-motile/biofilm state in bacteria is the bacterial second messenger c-di-GMP . This mo20–bolA morphogene and the signaling molecule c-di-GMP. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in E. coli.In this report, we have unravelled a direct connection between the E. coli to develop biofilms  strain and compared with the swimming capacity of bolA deletion (ΔbolA) mutant and bolA-overexpressing strains. The overexpression was obtained with a plasmid expressing bolA either under the control of its own promoters (bolA+) or under the control of an arabinose-inducible promoter (bolA++). In bolA deletion mutant . Despite this fact, a bolA ycgR double mutant was constructed and the swimming ability of E. coli was analyzed. If the effect on swimming occurred via YcgR, even by an indirect pathway, we would expect to observe a reversion of the ΔbolA strain phenotype. However, our results were inconclusive and it was not possible to clarify the influence of BolA in the YcgR-dependent flagellar rotation mechanism.In bacteria, YcgR reduces motility by interacting directly with flagellar motor proteins, slowing down flagellar rotor speed and altering the frequency of the rotational switch –31. The –bolA was overexpressed using the plasmid with its own promoters (bolA+), no difference in c-di-GMP levels in comparison to the wt strain levels was observed. However, the bolA++ strain showed a decrease in c-di-GMP levels of about 2-fold in comparison to the wt strain. Finally, in the bolA deletion mutant, the c-di-GMP levels in the cells increased 2-fold relative to the levels seen with the wt strain. This observation supports the data corresponding to the motility impairment previously detected in this strain; furthermore, it links BolA with the regulation of the c-di-GMP metabolite in bacteria for the first time.The ability of bacteria to regulate the planktonic-to-sessile lifestyle transition is also well known to involve the bacterial second messenger c-di-GMP , 25. ThiE. coli were previously studied (bolA strain versus bolA++ strain) were validated by (semi)quantitative reverse transcription PCR (RT-PCR)  presenting a difference representing an increase of more than 3.3-fold in the mRNA level in comparison to the ΔbolA strain results. Additionally, yhjH, encoding the PDE involved in the regulation of the mechanism of flagellar motor rotation  . All genrotation , 34, wasrotation , 35. On ΔbolA and bolA++ strains, respectively, could be due to the differential levels of expression of these enzymes. Both proteins were previously described as participating in an important cascade of c-di-GMP regulation  reduction in the levels of c-di-GMP in comparison to that seen with the single ΔbolA mutant  was influenced by BolA, we were interested in evaluating whether the elevated and reduced levels of c-di-GMP in the gulation . To testH strain  and C. IA mutant . Moreovebacteria , as expec-di-GMP . Howeverbacteria . Given tservable .10.1128/mBio.00443-17.1FIG S1 ydaM yhjH knockout mutant. (A) The bolA ydaM double mutant showed a phenotype similar to that of the bolA single mutant. (B) In the bolA++ strain lacking the yhjH gene, due to the high levels of BolA that were present simultaneously with the increased levels of c-di-GMP, a cumulative effect on motility impairment was observed. All plates were incubated at 37°C, and photographs were taken using ImageScanner III . Statistically significant differences were determined by measuring the diameter of the swimming halo. ****, P < 0.0001 (one-way ANOVA test). Download FIG S1, TIF file, 1.3 MB.Analysis of BolA effect on the motility of a Copyright © 2017 Moreira et al.2017Moreira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the bolA, and bolA++ strains were compared and verified to have phenotypes similar to those described in the literature. A knockout (ΔbolA) mutant was previously shown to be impaired in biofilm formation in comparison to a wt strain . Download FIG S2, TIF file, 1.2 MB.Analysis of BolA-dependent c-di-GMP regulation in biofilm formation. Biofilm biomass was estimated by the crystal violet method after 24 h of growth. **, Copyright © 2017 Moreira et al.2017Moreira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In conclusion, BolA not only contributes directly to the motility of bacteria  but alsoE. coli BolA to directly interact in vivo and in vitro with DNA molecules, influencing the rate of transcription of a given gene . Together, our data suggest that BolA plays an important role in the regulation of the enzymes responsible for the synthesis and degradation of the c-di-GMP signaling molecule.Previous results showed the ability of ven gene , 38, 39.ven gene . The resDifferent types of c-di-GMP effector proteins with downstream consequences were previously unravelled with respect to gene regulation in the cell  (bolA under the regulation of its own promoters (strain bolA+). However, when bacteria were in the stationary phase    and B. IqRT-PCR) , there wry phase , there wout 40%) . This shyhjH and, consequently, with the increase of c-di-GMP levels, expression of bolA was reduced  that are BolA dependent and that have been reported to be important in c-di-GMP regulation. As c-di-GMP is highly produced only during the transition from the exponential phase to the stationary phase, we performed the qRT-PCR analyses only in the stationary-phase cells for this set of samples. Similarly to what was observed before, in the absence of  reduced . Even ifpt level .E. coli by regulating the expression of DGCs and PDEs but also that the c-di-GMP signaling molecule is involved in the control of bolA expression. This cross-regulation seems to indicate that bacteria need to balance the amounts of these two components in order to react properly under stress conditions.Together, our results suggest not only that BolA is modulating the levels of c-di-GMP in The molecular mechanism behind the bacterial transition from the planktonic lifestyle to the biofilm lifestyle has been a fascinating object of study. Despite significant advances in the topic, there are still several interconnected pathways that need to be clarified. Here, we are adding an important piece to the puzzle by showing that BolA can interfere with this transition by balancing the intracellular concentration of the bacterial second messenger c-di-GMP.bolA strain.Recently, we showed that BolA is a very important transcriptional switch, that is specifically involved in the transition between the planktonic stage and the attachment stage of biofilm formation processes . ElevatebolA cells. In fact, the reduced swimming seen with this strain might be associated with the 1.7-fold-increased levels of c-di-GMP.The ability of bacteria to regulate the planktonic-to-sessile lifestyle transition is well known to involve the bacterial second messenger c-di-GMP , 25. ItsydaM DGC and yhjH PDE. Both genes encode proteins that have been previously described as participating in the cascade of c-di-GMP regulation linked by the YciR trigger enzyme  into SalI and SmaI sites of vector pSP417. The resulting plasmid was transformed in MC1061 ΔbolA and MC1061 competent cells to obtain strains CMA812 and CMA813, respectively. The λRed-mediated mutagenesis method (ydaM (CMA815) and yhjH (CMA817) deletion strains. Briefly, for the ΔydaM and ΔyhjH strains, a PCR fragment was obtained by amplification of pKD3 plasmid using primer pairs RNM236/237 and yhjHKOFor/yhjHKORev, respectively. The resulting fragments were transformed in MG1655 competent cells to allow recombination with the bacterial chromosome. P1 transduction was used to obtain strains CMA814, CMA816, and CMA818. CMA819 was constructed by transforming strain CMA816 with pCDA02 plasmid.All strains and plasmids used in this study are listed in s method  was used10.1128/mBio.00443-17.3TABLE S1 TABLE S1, DOCX file, 0.02 MB.Strains and plasmids used in this work. Download Copyright © 2017 Moreira et al.2017Moreira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00443-17.4TABLE S2 TABLE S2, DOCX file, 0.1 MB.Oligonucleotides used in this work. Download Copyright © 2017 Moreira et al.2017Moreira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the All constructs were confirmed by DNA sequencing at STAB Vida, Portugal.E. coli strains were grown in Luria-Bertani broth (LB) at 37°C with agitation, unless differently specified. When appropriate, antibiotics were used at the following concentrations: 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 50 µg/ml chloramphenicol.E. coli BL21(DE3) strain. Purification of BolA was performed by histidine affinity chromatography using HisTrap chelating HP columns  and an AKTA fast protein liquid chromatography system .BolA overexpression was performed using the pPFA02 plasmid and protein purification as previously described . The plaMotility assays were performed as previously described . When apExtraction and quantification of c-di-GMP were performed using bacterial liquid cultures obtained in the exponential and stationary phases, according to the method of Spangler et al. . The proE. coli strains grown overnight in LB and diluted to a final optical density at 600 nm (OD600) of 0.1 in fresh LB media and incubated at 37°C. Once the early stationary phase was reached, the cultures were diluted to a final OD600 of 0.1 in M63 media supplemented with 0.2% of arabinose. Aliquots (200 µl) were transferred to a 96-well polyvinyl chloride (PVC) plate and incubated at 37°C without agitation for 24 h. The planktonic state was determined by measuring the OD600 of the unattached cells using a SpectraMax Plus 384 microplate reader (Molecular Devices). PVC plates were washed twice with Milli-Q (MQ) water and the attached bacteria stained with 0.1% crystal violet for 10 min. The crystal violet was solubilized by a solution of acetone-ethanol (1:4) and biofilm thickness estimated by measuring the OD570.Crystal violet biofilm assays were performed as previously described by Dressaire et al.  with theDifferentially regulated genes were identified as previously described . AnalysiTotal RNA extraction was performed as previously described . For qRTydeH, yahA, yhjH, yhjK, and ydaM expression, respectively. As an independent control, 16S rRNA was amplified with specific primers 16srrnF/16srrnR.RT-PCR reactions were performed as previously described . Primer bolA mRNA was determined with a Rotor-Gene 3000 system (Corbett), using a SensiFast SYBR kit (Bioline) according to the supplier’s instructions. When necessary, bacterial cultures were supplemented with 1 μM c-di-GMP. qRT-PCR was performed with bolA gene-specific primers (RNM115 and RNM116). The cysG housekeeping gene was used as a control (RNM125 and RNM126). Relative quantifications of gene expression were calculated by the threshold cycle (ΔΔCT) method (The expression of ) method .KD of the interaction of BolA with the substrates was determined using a BLItz system (ForteBio Inc.) according to the instructions of the manufacturer. Biotin-labeled DNA fragments were obtained by PCR, using RNM164*/RNM195, RNM196/RNM197*, RNM198*/RNM199, and RNM200*/RNM201 primers to amplify ydaM, yhjH, yhjK, and ydeH promoter regions, respectively. A negative-control experiment was performed with bovine serum albumin (BSA) protein and flhDC promoter sequence.The 420 − (1.75 × OD550)]/(t × v × OD600) (where t represents time and v represents volume).The β-galactosidase activity of pRMA03 transcriptional fusion  was assaGSE58509) were accessed from the Gene Expression Omnibus (GEO) (Microarray raw data (GenBank accession number us (GEO) .